Structural analysis of effector functions related motifs, complement activation and hemagglutinating activities in Lama glama heavy chain antibodies

Heavy chain antibodies (HCAbs), devoid of the light chains and the CH(1) domain, are present in the serum of camelids. IgG(2) and IgG(3) are HCAbs; whereas IgG(1) has the conventional structure. In order to study the immunological properties of llama HCAbs, from which to date little is known, llamas (Lama glama) HCAbs cDNA were cloned, sequenced and compared with other mammalian Igs. The sequence analysis showed that llama HCAbs cDNA organization is similar to other mammalian Igs and the presence of conserved binding motifs to Protein A, Protein G, FcγRI, FcγRIII and C1q in HCAbs were observed. In a previous work, different IgG isotypes purified by Protein A and Protein G chromatography, were assayed for their ability to fix complement. Both IgG(1) and the total serum were able to fix complement, whereas IgG(2) and IgG(3) fixed complement even in the absence of antigen (anti-complementary activity). Therefore, in this work we performed the complement activating activity of the different IgG isotypes purified under physiological conditions using Sephadex G-150 and their ability to induce hemagglutination. Llamas were immunized with sheep red blood cells (RBC) stroma and the different isotypes were purified from sera. Whole serum and IgG(1) could activate complement; however, HCAbs (IgG(2)+IgG(3)) could not, despite the presence of the C1q binding motif in their primary sequence. Unlike IgG(1), the fraction corresponding to IgG(2)+IgG(3) did not display hemagglutinating activity. Our findings suggest that HCAbs cannot crosslink efficiently with different antigens and that the C1q binding site might be hindered by the proximity of the variable domains.     

Immunobiological role of llama heavy-chain antibodies against a bacterial beta-lactamase

In 1993, a fraction of antibodies (Abs) devoid of L chain was found naturally occurring in the Camelidae. They were found to lack L chains, as well as the first constant heavy-chain domain (CH(1)) and therefore they were named "heavy-chain Abs" (HCAbs). Subsequent studies focused on the functional, structural and biochemical properties of recombinant variable fragments (rVHHs) of HCAbs. It was stated that rVHHs have an augmented capacity to interact with "partially hidden" epitopes, like enzymes active sites, and have an increased stability to thermal and chemical aggression. It has been suggested that these unconventional Abs could represent an evolutionary advantage, being more efficient than conventional Abs to inhibit microbial enzymes, and thus exerting a more protective immune response against pathogens. The present work focuses on the immunobiological role of HCAbs, in their capacity to inhibit microbial enzymes. Two animal models were selected, comprising a model for common vertebrates without HCAbs (rabbits), and a model for vertebrates with both conventional and unconventional Abs (Lama glama). A recombinant bacterial beta-lactamase (CTX-M-2) was selected as the microbial enzymatic antigen. After conventional immunization schedules, neither serum titers nor serum inhibitory capacity showed significant differences when rabbits and llamas were compared. These results indicate that the a priori assumption that the adaptive immune system of camelids could be better "prepared" to respond to bacterial enzymes because of the presence of HCAbs, is not always accurate. Furthermore, when the different llama antibody isotypes and subclasses were purified, it was demonstrated that the inhibitory capacity of total serum was due exclusively to IgG(1). HCAbs not only failed to inhibit CTX-M-2, but instead they activated its enzymatic activity. Altogether, these results indicate that the hypotheses extrapolated from the rVHHs properties need to be revised; the real role of HCAbs in vivo remains unknown, as well as their evolutionary cause.     

Immunoglobulin levels in tears and aqueous humor of horses before and after diethylcarbamazine (DEC) therapy

A quantitative investigation of equine tear and aqueous humor immunoglobulins was done using normal horses and ponies as well as horses and ponies infected with Onchocerca cervicalis. The equine immunoglobulin isotypes IgGa, IgM, IgA and IgG(T) were quantitated by either single radial immunodiffusion (SRID) or radioimmunoassay (RIA). Tear immunoglobulin levels for IgGa (128 +/- 151 micrograms/ml), IgA (1,664 +/- 1,038 micrograms/ml) and IgM (106 +/- 74 micrograms/ml) were measured, while IgG(T) was not detectable. In horses with ocular inflammation the IgGa was 18-fold higher in the tears, 2,269 +/- 3,077 micrograms/ml. Aqueous humor obtained by paracentesis of the normal equine eye under anaesthesia, resulted in values for IgGa (45.2 +/- 20.0 micrograms/ml), IgG(T) (5.2 +/- 2.0 micrograms/ml), IgM (1.3 +/- 4.8 micrograms/ml) and IgA (0.8 +/- 1.0 micrograms/ml). A pooled sample of normal aqueous fluid obtained from over 100 horses at an equine abbatoir in Indiana gave values of 1,150 micrograms/ml for IgGa, 65 micrograms/ml for IgG(T), 2.5 micrograms/ml for IgA and 3.0 micrograms/ml for IgM. In animals infected with 0. cervicalis and treated with Diethylcarbamazine (DEC), there was a marked elevation of IgGa and IgG(T) in the tears and aqueous humor while IgA and IgG(T) were also elevated slightly in the aqueous. The findings of elevated immunoglobulin isotypes in the aqueous humor may not be related to the DEC treatment and 0. cervicalis infections but rather to repeated paracentesis and the development of acute inflammation of the equine eye as a result of the trauma of paracentesis. The elevations in equine immunoglobulin isotypes in the tears after DEC treatment are not subject to the same caveat. The preferential elevation in IgGa and IgG(T) in the tears precedes the development of corneal opacities observed in the same horses. The concentration of specific antimicrofilarial antibody in these tears remains to be determined but may well account for a major share of the total immunoglobulins detected.     

Monoclonal antibodies against porcine immunoglobulin isotypes

Monoclonal antibodies (MCAs) against porcine immunoglobulin isotypes* G, G1, G2, M and A have been produced and characterized in detail. Epitope analysis using a competitive direct enzyme-linked immunosorbent assay (ELISA) indicated that the MCAs recognized 3 class-specific epitopes of IgG, 4 epitopes specific for IgG1, 3 epitopes specific for IgG2, 2 epitopes of IgM and 2 epitopes of IgA. Two MCAs against IgG2 were shown to react with an allotypic determinant (B2) and one MCA against IgM is probably allotype specific. The production of MCAs specific for IgG and for its subclasses G1 and G2 and, in addition, the one-step isolation of nearly pure IgG1 and IgG2 preparations by immunoaffinity chromatography using MCA 34.1.1a (anti-IgG2) confirmed the existence of at least two subclasses of IgG. Preliminary results further suggested the existence of a subpopulation of IgG1 which could be eluted selectively from Protein A-Sepharose columns at pH 5.0. MCA 34.17.2a appeared to react preferentially with this IgG1 subpopulation and could be used to isolate a similar IgG1 subpopulation by immuno-affinity chromatography. Several of the MCAs have been successfully applied for the detection of porcine immunoglobulin isotypes by a double antibody sandwich ELISA and for the (isotype-specific) detection of antibodies against various porcine viruses. The availability of a full set of MCAs against porcine immunoglobulin isotypes will stimulate and facilitate the further study of the porcine immune system.     

Mechanism and isotypes involved in passive immunoglobulin transfer to the newborn alpaca (Lama pacos)

Crias, newborn alpacas (Lama pacos), that were almost agammaglobulinemic at birth had a 70% increase in total serum proteins within 24 hours largely because of absorption of gamma globulins from colostrum. Immunoglobulin G was the isotype in highest concentration in colostrum and in serum from 24-hour-old crias. The serum IgG concentration of 10 crias increased linearly (r = 0.97) from a mean of 0.3 mg/ml (+/- 0.1 SD) for serum collected before crias suckled to a maximal mean of 30.1 mg/ml (+/- 8.1 SD) at 24 hours. The 24-hour concentration decreased by half in 10 days. Immunoglobulin M also was absorbed from colostrum and increased linearly (r = 0.99) from a mean of 0.5 mg/ml (+/- 0.1 SD) for serum collected before crias suckled to a maximal mean of 4.2 mg/ml (+/- 2.2 SD) 24 hours after birth. The 24-hour serum concentration of IgM decreased by half in 7 days. Therefore, on a weight basis, 7 times more IgG than IgM was transferred to crias; IgG accounted for greater than 85% of the passively transferred proteins in serum of 24-hour-old crias. Absorption of functional antibodies of IgG and IgM isotypes from colostrum of immunized dams by crias also was demonstrated. Immunoglobulin G and IgM antibody titers to chicken RBC increased linearly to maximal geometric mean titers of 1,139 and 843, respectively, 24 hours after birth. The 24-hour IgG and IgM antibody titers decreased by half in 6 and 3.8 days, respectively. Purified alpaca IgG had a molecular mass of 166 kilodaltons, a predominant gamma mobility, and an extinction coefficient of 14.1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antibody dynamics in BRSV-infected Danish dairy herds as determined by isotype-specific immunoglobulins

Using specific ELISAs, antibody levels of four different isotypes to bovine respiratory syncytial virus (BRSV) were determined in calves, following experimental BRSV infection.Most calves experienced an increase in the specific IgM and IgG1 titres about 6-10 days after infection with BRSV. The IgM titre was transient showing positive titres for only 5-10 days, while specific IgG1 was present for a longer time. IgA was detected concomitantly with IgM but at a lower level. Production of IgG2 anti-BRSV antibodies was detected from 3 weeks after infection.In two closed herds, repeated blood samplings were performed on young stock to analyse maternal immunity. The passively transferred antibodies were mainly of the IgG1 isotype and the half-life of IgG1 to BRSV was estimated to be 26.6 days. One of the herds had an outbreak of enzootic pneumonia, diagnosed to be caused by BRSV. Furthermore, another herd with acute BRSV was followed by weekly blood samples in six calves; in both herds IgM and IgG1 was detected shortly after the appearance of clinical signs. Serum samples from 50 Danish dairy herds (453 samples) were tested for immunoglobulins of the isotypes IgG1, IgG2 and IgM. The presence of antibodies to BRSV was widespread and more than 54% of the samples had BRSV antibodies of both the IgG1 and IgG2 isotypes indicating a high herd prevalence to BRSV. Test samples from two herds out of 50 were free from all isotypes to BRSV.     

Usefulness of serological examinations in the analysis of Salmonella infections in pig herds

The development of the antibody concentration against lipopolysaccharide (LPS) of S. Typhimurium und S. Choleraesuis in rearing pigs during the fattening period and in breeding sows of the corresponding age was recorded. The studies revealed the following results. Antibodies of isotypes IgG1 and IgG2 revealed a more pronounced specificity against the according Salmonella serovar than IgM antibodies. The calculated "antibody percent value" based on the total amount of Salmonella antibodies is mainly determined by the IgM antibodies in sera and meat juice, respectively. In fattening pigs a significant increase of antibodies against IgM and total Ig was observed between week 3 and 10 after beginning of the rearing period. In breeding pigs this increase was detectable already earlier. In only 3 out of 10 groups an increase of IgG1 and IgG2 was also seen. The detected significant increase of total Ig and IgM in the other groups might be the result of a less intensive exposure to salmonellas or it might be due to an increase of unspecific antibodies induced by other antigens. Serological investigations represent a valuable tool to record the intensity and development in time of the Salmonella exposure in pigs farms. Examination of total Ig is an appropriate method to detect pig herds with a high level of Salmonella exposure, for detailed epidemiological studies in pig farms the examination of antibody isotypes will give more comprehensive information.     

Dynamics of Leishmania-specific immunoglobulin isotypes in dogs with clinical leishmaniasis before and after treatment

Concentrations of Leishmania-specific immunoglobulin G (IgG), immunoglobulin M (IgM), and immunoglobulin A (IgA) isotypes were analyzed by enzyme-linked immunosorbent assay (ELISA) in 23 dogs naturally infected with Leishmania infantum before and 1 year after initiating drug therapy. Results showed a high expression and prevalence of Leishmania-specific IgG (176.4 +/- 89 ELISA units [EU]), IgM (105.3 +/- 95.5 EU), and IgA (153.6 +/- 98 EU) in dogs before treatment (median +/- interquartile range EU). One year after treatment was started, dogs were classified as responsive dogs (RDs; n = 13) or unresponsive dogs (UDs; n = 10) based on clinicopathologic findings. Both groups of dogs experienced a statistically significant decrease (P < .05) in Leishmania-specific IgG (RDs = 27%, UDs = 41%), IgM (RDs = 42%, UDs = 29%), and IgA (RDs = 56%, UDs = 46%). Concentrations of specific IgG and IgM were not different at diagnosis or after treatment between the 2 groups. However, the median value for Leishmania-specific IgA 1 year after treatment was significantly lower (P < .05) in RDs (60.8 +/- 67 EU) than in UDs (117 +/- 54 EU). Examination of our data indicates that both the IgA isotype, which is mostly produced by mucosal plasma cells, and the IgM isotype are increased in infected symptomatic dogs, as previously reported for IgG. These 3 isotypes decreased significantly 1 year after initiation of medical treatment.     

Control of immunoglobulin isotype production by porcine B-cells cultured with cytokines

Cytokines regulate immunoglobulin (Ig) isotype production following the Th1/Th2 paradigm, derived from studies of inbred mice. In pigs, it is not known which, if any, Ig isotypes may reflect a Th1/Th2 response. To evaluate this, purified porcine CD21(+) B-cells were co-cultured with Staphylococcus aureus Cowan strain 1 or Escherichia coli lipopolysaccharide as B-cell mitogens together with recombinant human IL-2, and recombinant porcine (rp) interferon (IFN)-gamma, IL-12 or IL-10. While the mitogens increased B-cell proliferation, cytokines had no additional effect. A quantitative competitive enzyme-immuno assay was used to measure concentrations of porcine IgM, IgG(1) and IgG(2) in B-cell culture supernatants. In vitro, porcine B-cells produced IgG(2), 106 +/- 17.3 microg/ml; IgG(1) 107 +/- 38.3 microg/ml and IgM 25.6 +/- 8.45 microg/ml. In some individuals, Th1 cytokines such as rpIFN-gamma and IL-12, enhanced IgG(2) in the face of low concentrations of IgG(1). Furthermore, individual responses, in some cases, tended to be diametrically opposed, reminiscent of previously documented categorical immune responses in pigs such that some individuals produced high concentrations of IgG(1) in response to the various doses of rp cytokines, while others produced lower concentrations. Pigs may generate a high IgG(1):IgG(2) ratio in response to rpIL-10, and possibly to other Th2-associated cytokines. However, B-cell response to rp cytokines in vitro exhibits marked variation by pig, a feature that is likely a function of highly variable individual genotypes and their interaction with complex environments.     

Isotype-specific ELISAs for the detection of antibodies to bovine respiratory syncytial virus

Isotype-specific ELISAs for the detection of antibodies to bovine respiratory syncytial virus (BRSV) are described. BRSV-specific IgG1 and IgG2 were determined in indirect double antibody sandwich assays. For IgA and IgM antibody capture assays were used. The isotype specificity of the assays was confirmed by the observation that samples with a high titre of BRSV-specific antibodies of particular isotype were negative in the assays for the other isotypes and vice versa. Comparison of the results obtained in the ELISAs and in the virus neutralisation test showed that acute phase antibodies were more efficiently detected in the latter. It also showed that the presence of BRSV-specific IgA was not correlated with neutralising activity in vitro. The serum antibody response of BRSV-infected seronegative calves from the field consisted of a nearly simultaneous increase of IgM, IgA and IgG1-antibodies in the acute phase of the disease, while the IgG2-response followed at various intervals thereafter. In young animals with maternal antibodies a different pattern was found. There was no increase in IgG1 and IgG2, but six of eight animals showed a weak IgM response and two of these six calves also showed a weak and short lasting IgA response. Because maternal antibodies are insufficiently effective in protecting calves against BRSV, the presence of such antibodies at mucosal surfaces was investigated. Maternal immunity was found to be restricted to IgG1 antibodies in serum. This agrees with the failure of maternal antibodies to protect mucosal surfaces against BRSV infection.     

Serum antibody response and Cryptosporidium parvum oocyst antigens recognized by sera from naturally infected sheep.

The response of specific serum immunoglobulins (IgG, IgM and IgA) and the major antigens of Cryptosporidium parvum recognized by these isotypes were investigated by using enzyme-linked immunosorbent assay and immunoblot techniques in lambs and ewes naturally infected throughout an outbreak of cryptosporidiosis. Serum samples were collected from 20 lambs the first day they showed diarrhoea (D1), and Days 11 and 22, in addition to single serum samples from 17 of their dams. Serum anti-C. parvum IgG, IgM and/or IgA antibodies were detected in lambs as early as Day 1. Levels of IgM antibodies remained steady from D1 to D11 and increased at D22, whereas the IgG response decreased from D1 to D11 and subsequently increased. In contrast, IgA antibodies rapidly fell from D1 and all lambs were seronegative at D11 and D22. The highest levels of specific antibodies were detected in sera from ewes. In fact, all ewes were seropositives for IgM and IgA isotypes and most (16/17) showed positive levels of IgG. Four protein fractions (37-39, 42-48, 51-57 and 60-69 kDa) were the most frequently recognized by IgG and IgM from lamb sera. A low molecular weight fraction (12-14 kDa) reacting with IgG and IgA in most lamb sera was scarcely recognized by IgM and three broad bands were frequently recognized by IgA antibodies (23-25, 51-57 and 90-95 kDa). The recognition pattern of 23-25 kDa peptides by IgA from lamb sera clearly increased with the age. Peptides of 42-48, 51-57, 60-69 and 71-78 kDa were most frequently recognized by IgG and IgM from ewe sera. In relation to IgA antibodies from ewe sera, a frequent immunoreactivity was found with proteins in the intervals between 12 and 22 kDa as well as between 32 and 34 kDa and practically all sera reacted with fractions from 42 to 95 kDa.     

Colostral volume and immunoglobulin G and M determinations in mares

Colostral volume and IgG and IgM concentrations were determined in 6 multiparous mares at foaling and them every 2 hours from 16 to 20 hours after parturition. Serum IgG and IgM concentrations at foaling also were determined in each mare. The rate of mammary secretion was 292 +/- 26 ml/h (range, 202 to 389 ml/h), and the colostral volume was 5.1 +/- 0.5 L (range, 3.2 to 7.0 L). The colostral IgG and IgM contents were 440 +/- 106 g (range, 199 to 855 g) and 3.1 +/- 0.9 g (range, 0.7 g to 7.1 g), respectively. There was no significant correlation between serum and initial colostral IgG and IgM concentration or between serum and total colostral IgG or IgM values. The colostral IgG and IgM concentrations at foaling correlated well with the total colostral IgG and IgM contents, respectively. The initial 250 ml of colostrum contained 10 +/- 1.4% (range, 6.0 to 13.9%) and 6 +/- 1.0% (range, 2.4 to 8.5%) of the total IgG and IgM contents, respectively, and the initial 500 ml of colostrum contained 20 +/- 2.7% (range 12.0 to 27.1%) and 14 +/- 1.2% (8.2 to 17%) of the total colostral IgG and IgM contents, respectively.     

Quantitation of bovine immunoglobulins in culture fluids by use of sandwich radioimmunoassay with monoclonal antibodies

Bovine immunoglobulin isotype-specific murine monoclonal antibodies were used in sandwich radioimmunoassays to detect and quantitate bovine IgG1, IgG2, IgM, and IgA in culture fluids. The concentrations of bovine immunoglobulins in unknown samples were extrapolated from standard curves generated with bovine monoclonal immunoglobulins. The lowest detection limits for the bovine immunoglobulin isotypes ranged from 65 to 270 ng/ml.     

Intestinal antibody response after vaccination and infection with rotavirus of calves fed colostrum with or without rotavirus antibody

The intestinal and systemic antibody response of calves vaccinated and/or challenged with rotavirus was studied employing isotype-specific ELISAs for the detection of IgG1, IgG2, IgM and IgA antibodies to rotavirus. Monoclonal antibodies to bovine immunoglobulin isotypes of proven specificity were used as conjugated or catching antibody. Five days after oral inoculation (dpi) of a 5-day-old gnotobiotic calf with rotavirus, IgM rotavirus antibodies were excreted in faeces, followed 5 days later by IgA rotavirus antibodies. The increase in IgM rotavirus antibody titre coincided with the inability to detect further rotavirus excretion. Faeces IgM and IgA rotavirus antibody titres fell to low levels within 3 weeks post infection. IgG1 and IgG2 rotavirus antibodies were not detected in faecal samples. In serum, antibodies to rotavirus of all four isotypes were detected, starting with IgM at 5 dpi. Two SPF-calves, which were fed colostrum free of rotavirus antibodies, were vaccinated with a modified live rotavirus vaccine and challenged with virulent rotavirus 6 days later. Upon vaccination, the calves showed an antibody response similar to the response of the infected gnotobiotic calf. Intestinal IgM rotavirus antibodies were excreted before or on the day of challenge and appeared to be associated with protection against challenge infection with virulent virus and rotavirus-induced diarrhoea. In 3 control calves, which were challenged only, the antibody patterns also resembled that of the gnotobiotic calf and again the appearance of IgM rotavirus antibodies coincided with the end of the rotavirus detection period. Two other groups of 3 SPF-calves were treated similarly, but the calves were fed colostrum with rotavirus antibodies during the first 48 h of life. These calves excreted passively acquired IgG1 and IgG2 rotavirus antibodies in their faeces from 2 to 6 days after birth. After vaccination, no IgM or IgA antibody activity in serum or faeces was detectable. Upon challenge, all calves developed diarrhoea and excreted rotavirus. Seven to 10 days after challenge low levels of IgM rotavirus antibody were detected for a short period. These data indicate that the intestinal antibody response of young calves to an enteric viral infection is associated with the excretion of IgM antibodies, immediately followed by IgA antibodies. This response is absent or diminished in calves with passively acquired specific antibodies which may explain the failure to induce a protective intestinal immune response by oral vaccination with modified live rotavirus of calves fed colostrum containing rotavirus antibodies.     

Distribution of immunoglobulin isotypes and Salmonella antibodies in blood serum and meat juice of pigs

Using the close linear regression between the logarithm of the dilution degree of a sample and the logarithm of the extinction measured in an ELISA both the relative concentrations of immunoglobulines of the isotypes IgG, IgM and IgA and of the LPS antibodies against S. Typhimurium of the different isotypes in blood sera and meat juice of 15 slaughtered pigs were detected and compared. Furthermore the total concentration of antibodies against LPS of S. Typhimurium according to the "meat juice ELISA" were compared. Distribution of immunoglobulines between serum and meat juice revealed individual differences between the animals as well as between the different immunoglobulin-isotypes. Within the same isotype the ratio of the concentrations of anti-LPS Salmonella Typhimurium antibodies between serum and meat juice was significantly closer than relating the whole of immunoglobulines of the referred isotype. In order to detect pig herds with a high level of Salmonella exposure a comparison of the 1:30 diluted meat juice samples with the 1:400 diluted blood sera is justified, however, for detailed epidemiological or scientific studies there is a need to consider the existing differences between the immunoglobuline-isotypes as well as between the specificity of antibodies and of total immunoglobulines. While the concentration of Salmonella antibodies of the isotypes IgG1, IgG2 and IgA showed a clear and statistically significant correlation between both one below the other and with the total amount of Salmonella antibodies, this connection could not be established for the total amount of immunoglobulines of different isotypes and the IgM-antibodies.     

Passive transfer of colostral immunoglobulin G in neonatal llamas and alpacas

OBJECTIVE: To evaluate precolostral hypogammaglobulinemia in neonatal llamas and alpacas, to determine when postcolostral peak serum IgG concentrations develop, to determine whether differences in postcolostral serum IgG concentrations between llamas and alpacas exist, and to determine postcolostral half-life of serum IgG in llamas and alpacas. DESIGN: Prospective observational study. ANIMALS: 29 llama and 10 alpaca crias. PROCEDURE: Blood samples were collected prior to suckling and on days 1, 2, and 3 after parturition and analyzed for serum IgG concentration by use of a commercial radial immunodiffusion assay. Additional samples were collected on days 8, 13, and 18 from 8 crias to determine mean half-life of IgG. RESULTS: Llamas and alpacas are born severely hypogammaglobulinemic. Mean serum IgG concentrations for day-1, -2, and -3 samples for llamas were 1,578 mg/dl, 1,579 mg/dl, and 1,401 mg/dl, respectively, and for alpacas were 2,024 mg/dl, 1,806 mg/dl, and 1,669 mg/dl, respectively. Peak serum immunoglobulin concentration developed between days 1 and 2. Mean half-life of IgG for all crias was 15.7 days. CONCLUSIONS AND CLINICAL RELEVANCE: Although increased mortality has been linked to failure of passive transfer, it is clearly possible to raise crias that have low serum immunoglobulin concentrations. Llamas and alpacas do not differ significantly with respect to immunoglobulin absorption or IgG concentration in neonates. The optimal sampling time for passive transfer status is between 1 and 2 days.     

IgA, igG1, IgG2, IgM, and BSA in serum and mammary secretion throughout lactation

Bovine IgG1, IgG2, IgA, and IgM were measured in the serum and lacteal secretions of six cows from 10 days prepartum to 240 days of lactation. Immunoglobulins in lacteal secretions were expressed in units of concentration (mg/ml) as well as in total daily output. All isotypes were selectively accumulated during colostrum formation. The rate of IgG1 accumulation decreased rapidly after calving; this decrease corresponded to a return to normal serum levels of this immunoglobulin. Selective accumulation of IgA > IgM > IgG1 was maintained throughout lactation, but IgG2 showed no selective accumulation beyond 5 days postpartum. In serum, IgA and IgM levels were elevated at parturition and showed a significant decrease postpartum. Increases in serum IgA levels 60 days postpartum corresponded to a rise in lacteal concentration. The concentration of all immunoglobulins increased during late lactation, coincident with a major reduction in milk yield. Six strains of mastitis-causing organisms were cultured during the period of the experiment; however, none resulted in clinical mastitis or showed an effect on immunoglobulin secretion.     

Anti-erythrocyte natural antibody activity in the unconventional 'heavy chain' immunoglobulins of Indian desert camel (Camelus dromedarius)

Members of the family Camelidae contain unconventional 'heavy chain' antibodies (HCAbs) that are devoid of light chains (LCs) in their structure and occur under physiological conditions. The spectrum of antigenic specificities in HCAbs and hence their biological significance is not known at present. Recent studies have however indicated that they contribute significantly towards the immunoglobulin (Ig) receptor diversity. The present study was planned to investigate the natural antibody (NAb) activity in camel HCAbs as further indication of the wide spectrum of their antigenic specificities. Detection of NAbs in the sera and isolated HCAbs of Indian Thar desert camels was undertaken against erythrocyte antigens (E-Ags) from eleven animal species including nine mammals, chicken and frog by using direct haemagglutination (HA) and indirect Coombs' test. HCAbs were found to behave as 'incomplete antibodies' and agglutinated erythrocytes of different animal species in indirect Coombs' test using rabbit anti-camel IgG3 (HCAbs) antiserum. Variations were noticed in the Coombs' titres against erythrocytes from different species. HCAbs also reacted against E-Ags in immunoblots. These findings provide further evidence that camelid HCAbs are produced against diverse antigens (Ags) under natural conditions, thereby contributing to camelid Ig receptor diversity.     

Monoclonal precipitating antibodies to porcine immunoglobulin M

Fusion of splenic immunocytes from a porcine IgM-immunized BALB/c mouse with SP2/0 mouse myeloma cells resulted in 231 primary hybrids. Culture fluids of the primary hybrids were screened for antibody production by enzyme-linked immunosorbent assay (ELISA) against porcine IgM and by radial immunodiffusion versus porcine serum. Culture fluids of 10 of the primary hybrids were positive in IgM-ELISA and radial immunodiffusion. Six of these primary hybrids (1A11, 1D10, 2D7, 2E2, 3B11, and 5C9) were cloned, and ascitic fluids were produced using cloned primary hybrids. The monoclonal antibodies (Mabs) in ascitic fluids were characterized as to their reactivity with porcine immunoglobulin isotypes. All six Mabs had mouse IgG1, K isotype and were mu-chain specific as they formed single precipitin lines against porcine serum and porcine IgM and no lines against porcine IgG, IgA, and fetal porcine serum in immunodiffusion and immunoelectrophoresis. In indirect ELISA, all Mabs reacted with porcine serum, porcine IgM, and mu-chains but did not react with porcine IgG, IgA, or light chains. All six Mabs were species-specific and recognized either of two antigenic regions of mu-chain. These Mabs have been successfully used to detect IgM-containing cells in tissue sections, to detect IgM in serum, and to quantitate surface membrane IgM-bearing cells in peripheral blood.     

Maternally and naturally acquired antibodies to Mannheimia haemolytica and Pasteurella multocida in beef calves

The dynamics and duration of maternally derived antibodies as well as the onset of acquired immunity against Mannheimia haemolytica and Pasteurella multocida in range-pastured beef calves were investigated. Two groups of unvaccinated cattle were used in this study. Serum antibody responses were measured by enzyme-linked immunoassay for antibodies of the IgG1, IgG2 and IgM isotypes binding M. haemolytica whole cells (WC) or leukotoxin (LKT) and P. multocida outer membrane proteins (OMPs). Comparisons of mean antibody responses to M. haemolytica LKT and WC and P. multocida OMPs were made within each group. Maternally derived antibodies against M. haemolytica and P. multocida reached lowest levels at 30-90 days after birth. Calves began production of antibodies against M. haemolytica and P. multocida between 60 and 90 days of age in both groups. Based on the results of this study, in beef herds vaccinated against M. haemolytica and/or P. multocida, it may be best to vaccinate calves around 3 months of age. In contrast, beef calves from unvaccinated herds might benefit from vaccination at 4 months of age.